Method for treating amyloidosis

ABSTRACT

Therapeutic compounds and methods for inhibiting amyloid deposition in a subject, whatever its clinical setting, are described. Amyloid deposition is inhibited by the administration to a subject of an effective amount of a therapeutic compound comprising an anionic group and a carrier molecule, or a pharmaceutically acceptable salt thereof, such that an interaction between an amyloidogenic protein and a basement membrane constituent is inhibited. Preferred anionic groups are sulfonates and sulfates. Preferred carrier molecules include carbohydrates, polymers, peptides, peptide derivatives, aliphatic groups, alicyclic groups, heterocyclic groups, aromatic groups and combinations thereof.

RELATED APPLICATIONS

This Application is a continuation of application Ser. No. 11/316378filed on Dec. 22, 2005, which is a continuation of application Ser. No.10/777926 filed on Feb. 11, 2004, which is a continuation of applicationSer. No. 10/125063 filed on Apr. 18, 2002, which is a continuation ofapplication Ser. No. 09/780233 filed on Feb. 9, 2001, which is acontinuation of application Ser. No. 09/322577 filed on May 27, 1999,which is a continuation of application Ser. No. 08/463548 filed on Jun.5, 1995, now U.S. Pat. No. 5,972,328, which is a continuation-in-part ofapplication Ser. No. 08/403230 filed on Mar. 15, 1995, now U.S. Pat. No.5,643,562, which is a continuation-in-part of application Ser. No.08/315391 filed on Sep. 29, 1994, which is a continuation-in-part ofapplication Ser. No. 08/219798 filed on Mar. 29, 1994, which is acontinuation-in-part of application Ser. No. 08/037844 filed on Mar. 29,1993. This application is also related to application Ser. No.08/542,997, now U.S. Pat. No. 5,840,294, which is a continuation-in-partof Ser. Nos. 08/463,548, and 08/472,692, now U.S. Pat. No. 5,728,375,which is a continuation of Ser. No. 08/463,548. The contents of all ofthe above applications are incorporated herein by reference.

BACKGROUND OF INVENTION

Amyloidosis refers to a pathological condition characterized by thepresence of amyloid. Amyloid is a generic term referring to a group ofdiverse but specific extracellular protein deposits which are seen in anumber of different diseases. Though diverse in their occurrence, allamyloid deposits have common morphologic properties, stain with specificdyes (e.g., Congo red), and have a characteristic red-green birefringentappearance in polarized light after staining. They also share commonultrastructural features and common x-ray diffraction and infraredspectra.

Amyloidosis can be classified clinically as primary, secondary, familialand/or isolated. Primary amyloidosis appears de novo without anypreceding disorder. Secondary amyloidosis is that form which appears asa complication of a previously existing disorder. Familial amyloidosisis a genetically inherited form found in particular geographicpopulations. Isolated forms of amyloidosis are those that tend toinvolve a single organ system. Different amyloids are also characterizedby the type of protein present in the deposit. For example,neurodegenerative diseases such as scrapie, bovine spongiformencephalitis, Creutzfeldt-Jakob disease and the like are characterizedby the appearance and accumulation of a protease-resistant form of aprion protein (referred to as AScr or PrP-27) in the central nervoussystem. Similarly, Alzheimer's disease, another neurodegenerativedisorder, is characterized by congophilic angiopathy, neuritic plaquesand neurofibrillary tangles, all of which have the characteristics ofamyloids. In this case, the plaques and blood vessel amyloid is formedby the beta protein. Other systemic diseases such as adult-onsetdiabetes, complications of long-term hemodialysis and sequelae oflong-standing inflammation or plasma cell dyscrasias are characterizedby the accumulation of amyloids systemically. In each of these cases, adifferent amyloidogenic protein is involved in amyloid deposition.

Once these amyloids have formed, there is no known therapy or treatmentwhich significantly dissolves the deposits in situ which is widelyaccepted.

SUMMARY OF THE INVENTION

This invention provides methods and compositions which are useful in thetreatment of amyloidosis. The methods of the invention involveadministering to a subject a therapeutic compound which inhibits amyloiddeposition. Accordingly, the compositions and methods of the inventionare useful for inhibiting amyloidosis in disorders in which amyloiddeposition occurs. The methods of the invention can be usedtherapeutically to treat amyloidosis or can be used prophylactically ina subject susceptible to amyloidosis. The methods of the invention arebased, at least in part, on inhibiting an interaction between anamyloidogenic protein and a constituent of basement membrane to inhibitamyloid deposition. The constituent of basement membrane is aglycoprotein or proteoglycan, preferably heparan sulfate proteoglycan. Atherapeutic compound used in the method of the invention can interferewith binding of a basement membrane constituent to a target binding siteon an amyloidogenic protein, thereby inhibiting amyloid deposition.

In one embodiment, the method of the invention involves administering toa subject a therapeutic compound having at least one anionic groupcovalently attached to a carrier molecule which is capable of inhibitingan interaction between an amyloidogenic protein and a glycoprotein orproteoglycan constituent of a basement membrane to inhibit amyloiddeposition. In one embodiment, the anionic group covalently attached tothe carrier molecule is a sulfonate group. Accordingly, the therapeuticcompound can have the formula:Q-[—SO₃ ⁻X⁺]_(n)wherein Q is a carrier molecule; X⁺ is a cationic group; and n is aninteger. In another embodiment, the anionic group is a sulfate group.Accordingly, the therapeutic compound can have the formula:Q-[—SO₃ ⁻X⁺]_(n)wherein Q is a carrier molecule; X⁺ is a cationic group; and n is aninteger. Carrier molecules which can be used include carbohydrates,polymers, peptides, peptide derivatives, aliphatic groups, alicyclicgroups, heterocyclic groups, aromatic groups and combinations thereof.Preferred therapeutic compounds for use in the invention includepoly(vinylsulfonic acid), ethanesulfonic acid, sucrose octasulfate,1,2-ethanediol disulfuric acid, 1,2-ethanedisulfonic acid,1,3-propanediol disulfuric acid, 1,3-propanedisulfonic acid,1,4-butanediol disulfuric acid, 1,4-butanedisulfonic acid,1,5-pentanedisulfonic acid, taurine, 3-(N-morpholino)propanesulfonicacid, tetrahydrothiophene-1,1-dioxide-3,4-disulfonic acid,4-hydroxybutane-1-sulfonic acid, or pharmaceutically acceptable saltsthereof.

The therapeutic compounds of the invention are administered to a subjectby a route which is effective for inhibition of amyloid deposition.Suitable routes of administration include subcutaneous, intravenous andintraperitoneal injection. The therapeutic compounds of the inventionhave been found to be effective when administered orally. Accordingly, apreferred route of administration is oral administration. Thetherapeutic compounds can be administered with a pharmaceuticallyacceptable vehicle.

The invention further provides pharmaceutical compositions for treatingamyloidosis. The pharmaceutical compositions include a therapeuticcompound of the invention in an amount effective to inhibit amyloiddeposition and a pharmaceutically acceptable vehicle.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a bar graph illustrating the effect of poly(vinylsulfonatesodium salt) administered intraperitoneally on in vivo AA amyloiddeposition in mouse spleen.

FIG. 2 is a graph illustrating the effect of poly(vinylsulfonate sodiumsalt) on heparan sulfate proteoglycan binding to β-APP in tris-bufferedsaline (TBS).

FIG. 3 is a graph illustrating the effect of poly(vinylsulfonate sodiumsalt) on heparan sulfate proteoglycan binding to β-APP in phosphatebuffered saline (PBS).

FIG. 4 is a bar graph illustrating the effect of poly(vinylsulfonatesodium salt) administered orally on in vivo AA amyloid deposition inmouse spleen.

FIG. 5 is a graph illustrating the blood level of the amyloid precursor,SAA, over time for animals receiving poly(vinylsulfonate sodium salt)(open circles) and control animals (triangles).

FIG. 6 is a graph illustrating the effect of orally administeredpoly(vinylsulfonate sodium salt) on the course of AA amyloid depositionin mouse spleen when amyloid deposits were already present prior totreatment of the animals. The triangles represent the control animalsand the open circles represent the treated animals.

FIG. 7 is a graph illustrating the effect of orally administeredpoly(vinylsulfonate sodium salt) on splenic amyloid deposition when theinflammatory stimulus is maintained during the course of the experiment.The triangles represent the control animals and the open circlesrepresent the treated animals.

FIG. 8 is a graph illustrating the effect of orally administered ethanemonosulfonate, sodium salt (EMS) on in vivo AA splenic amyloiddeposition. The triangles represent the control animals, the opencircles represent animals receiving 2.5 mg/ml of EMS in their drinkingwater, and the open squares represent animals receiving 6 mg/ml of EMSin their drinking water.

FIGS. 9 and 10 depict the chemical structures of the WAS compoundsdescribed in Example 9.

DETAILED DESCRIPTION OF INVENTION

This invention pertains to methods and compositions useful for treatingamyloidosis. The methods of the invention involve administering to asubject a therapeutic compound which inhibits amyloid deposition.“Inhibition of amyloid deposition” is intended to encompass preventionof amyloid formation, inhibition of further amyloid deposition in asubject with ongoing amyloidosis and reduction of amyloid deposits in asubject with ongoing amyloidosis. Inhibition of amyloid deposition isdetermined relative to an untreated subject or relative to the treatedsubject prior to treatment. Amyloid deposition is inhibited byinhibiting an interaction between an amyloidogenic protein and aconstituent of basement membrane. “Basement membrane” refers to anextracellular matrix comprising glycoproteins and proteoglycans,including laminin, collagen type IV, fibronectin and heparan sulfateproteoglycan (HSPG). In one embodiment, amyloid deposition is inhibitedby interfering with an interaction between an amyloidogenic protein anda sulfated glycosaminoglycan such as HSPG. Sulfated glycosaminoglycansare known to be present in all types of amyloids (see Snow, A. D. et al.(1987) Lab. Invest. 56:120-123) and amyloid deposition and HSPGdeposition occur coincidentally in animal models of amyloidosis (seeSnow, A. D. et al. (1987) Lab. Invest. 56:665-675). In the methods ofthe invention, molecules which have a similar structure to a sulfatedglycosaminoglycan are used to inhibit an interaction between anamyloidogenic protein and basement membrane constituent. In particular,the therapeutic compounds of the invention comprise at least one sulfategroup or a functional equivalent thereof, for example a sulfonic acidgroup or other functionally equivalent anionic group, linked to acarrier molecule. In addition to functioning as a carrier for theanionic functionality, the carrier molecule can enable the compound totraverse biological membranes and to be biodistributed without excessiveor premature metabolism. Moreover, when multiple anionic functionalitiesare present on a carrier molecule, the carrier molecule serves to spacethe anionic groups in a correct geometric separation.

In one embodiment, the method of the invention includes administering tothe subject an effective amount of a therapeutic compound which has atleast one anionic group covalently attached to a carrier molecule. Thetherapeutic compound is capable of inhibiting an interaction between anamyloidogenic protein and a glycoprotein or proteoglycan constituent ofa basement membrane to thus inhibit amyloid deposition. The therapeuticcompound can have the formula:Q-[—Y⁻X⁺]_(n)wherein Y⁻ is an anionic group at physiological pH; Q is a carriermolecule; X⁺ is a cationic group; and n is an integer. The number ofanionic groups (“n”) is selected such that the biodistribution of thecompound for an intended target site is not prevented while maintainingactivity of the compound. For example, the number of anionic groups isnot so great as to inhibit traversal of an anatomical barrier, such as acell membrane, or entry across a physiological barrier, such as theblood-brain barrier, in situations where such properties are desired. Inone embodiment, n is an integer between 1 and 10. In another embodiment,n is an integer between 3 and 8.

An anionic group of a therapeutic compound of the invention is anegatively charged moiety that, when attached to a carrier molecule, caninhibit an interaction between an amyloidogenic protein and aglycoprotein or proteoglycan constituent of a basement membrane to thusinhibit amyloid deposition. For purposes of this invention, the anionicgroup is negatively charged at physiological pH. Preferably, the anionictherapeutic compound mimics the structure of a sulfated proteoglycan,i.e., is a sulfated compound or a functional equivalent thereof.“Functional equivalents” of sulfates are intended to includebioisosteres. Bioisosteres encompass both classical bioisostericequivalents and non-classical bioisosteric equivalents. Classical andnon-classical bioisosteres of sulfate groups are known in the art (seee.g. Silverman, R. B. The Organic Chemistry of Drug Design and DrugAction, Academic Press, Inc.: San Diego, Calif., 1992, pp. 19-23).Accordingly, a therapeutic compound of the invention can comprise atleast one anionic group including sulfonates, sulfates, phosphonates,phosphates, carboxylates, and heterocyclic groups of the followingformulas:

Depending on the carrier molecule, more than one anionic group can beattached thereto. When more than one anionic group is attached to acarrier molecule, the multiple anionic groups can be the same structuralgroup (e.g., all sulfonates) or, alternatively, a combination ofdifferent anionic groups can be used (e.g., sulfonates and sulfates,etc.).

The ability of a therapeutic compound of the invention to inhibit aninteraction between an amyloidogenic protein and a glycoprotein orproteoglycan constituent of a basement membrane can be assessed by an invitro binding assay, such as that described in the Exemplification or inU.S. Pat. No. 5,164,295 by Kisilevsky et al. Briefly, a solid supportsuch as a polystyrene microtiter plate is coated with an amyloidogenicprotein (e.g., serum amyloid A protein or β-amyloid precursor protein(β-APP)) and any residual hydrophobic surfaces are blocked. The coatedsolid support is incubated with various concentrations of a constituentof basement membrane, preferably HSPG, either in the presence or absenceof a compound to be tested. The solid support is washed extensively toremove unbound material. The binding of the basement membraneconstituent (e.g., HSPG) to the amyloidogenic protein (e.g., β-APP) isthen measured using an antibody directed against the basement membraneconstituent which is conjugated to a detectable substance (e.g., anenzyme, such as alkaline phosphatase) by detecting the detectablesubstance. A compound which inhibits an interaction between anamyloidogenic protein and a glycoprotein or proteoglycan constituent ofa basement membrane will reduce the amount of substance detected (e.g.,will inhibit the amount of enzyme activity detected).

Preferably, a therapeutic compound of the invention interacts with abinding site for a basement membrane glycoprotein or proteoglycan in anamyloidogenic protein and thereby inhibits the binding of theamyloidogenic protein to the basement membrane constituent. Basementmembrane glycoproteins and proteoglycans include laminin, collagen typeIV, fibronectin and heparan sulfate proteoglycan (HSPG). In a preferredembodiment, the therapeutic compound inhibits an interaction between anamyloidogenic protein and HSPG. Consensus binding site motifs for HSPGin amyloidogenic proteins have been described (see e.g. Cardin andWeintraub (1989) Arteriosclerosis 9:21-32). For example, an HSPGconsensus binding motif can be of the general formula X1-X2-Y-X3,wherein X1, X2 and X3 are basic amino acids (e.g., lysine or arginine)and Y is any amino acid. Modeling of the geometry of this site led todetermination of the following spacing between basic amino acid residues(carboxylate to carboxylate, in Angstroms):X1-X2 5.3±1.5 ÅX1-X3 7.1±1.5 ÅX2-X3 7.6±1.5 ÅThese values were determined using a combination of molecular mechanicsand semi-empirical quantum mechanics calculations. Molecular mechanicscalculations were performed using the MM2 force field equation.Semi-empirical molecular orbital calculations were performed using theAM1 Hamiltonian equation. The conformational space of the site wassampled using a combination of molecular dynamics (both high and lowtemperature) and Monte Carlo simulations.

Accordingly, in the therapeutic compounds of the invention, whenmultiple anionic groups are attached to a carrier molecule, the relativespacing of the anionic groups can be chosen such that the anionic groups(e.g., sulfonates) optimally interact with the basic residues within theHSPG binding site (thereby inhibiting interaction of HSPG with thesite). For example, anionic groups can be spaced approximately 5.3±1.5Å, 7.1±1.5 Å and/or 7.6±1.5 Å apart, or appropriate multiples thereof,such that the relative spacing of the anionic groups allows for optimalinteraction with a binding site for a basement membrane constituent(e.g., HSPG) in an amyloidogenic protein.

A therapeutic compound of the invention typically further comprises acounter cation (i.e., X⁺ in the general formula: Q-[—Y⁻X⁺]_(n)).Cationic groups include positively charged atoms and moieties. If thecationic group is hydrogen, H⁺, then the compound is considered an acid,e.g., ethanesulfonic acid. If hydrogen is replaced by a metal or itsequivalent, the compound is a salt of the acid. Pharmaceuticallyacceptable salts of the therapeutic compound are within the scope of theinvention. For example, X⁺ can be a pharmaceutically acceptable alkalimetal, alkaline earth, higher valency cation (e.g., aluminum salt),polycationic counter ion or ammonium. A preferred pharmaceuticallyacceptable salt is a sodium salt but other salts are also contemplatedwithin their pharmaceutically acceptable range.

Within the therapeutic compound, the anionic group(s) is covalentlyattached to a carrier molecule. Suitable carrier molecules includecarbohydrates, polymers, peptides, peptide derivatives, aliphaticgroups, alicyclic groups, heterocyclic groups, aromatic groups orcombinations thereof. A carrier molecule can be substituted, e.g. withone or more amino, nitro, halogen, thiol or hydroxy groups.

As used herein, the term “carbohydrate” is intended to includesubstituted and unsubstituted mono-, oligo-, and polysaccharides.Monosaccharides are simple sugars usually of the formula C₆H₁₂O₆ thatcan be combined to form oligosaccharides or polysaccharides.Monosaccharides include enantiomers and both the D and L stereoisomersof monosaccharides. Carbohydrates can have multiple anionic groupsattached to each monosaccharide moiety. For example, in sucroseoctasulfate, four sulfate groups are attached to each of the twomonosaccharide moieties.

As used herein, the term “polymer” is intended to include moleculesformed by the chemical union of two or more combining subunits calledmonomers. Monomers are molecules or compounds which usually containcarbon and are of relatively low molecular weight and simple structure.A monomer can be converted to a polymer by combination with itself orother similar molecules or compounds. A polymer may be composed of asingle identical repeating subunit or multiple different repeatingsubunits (copolymers). Polymers within the scope of this inventioninclude substituted and unsubstituted vinyl, acryl, styrene andcarbohydrate-derived polymers and copolymers and salts thereof. In oneembodiment, the polymer has a molecular weight of approximately 800-1000Daltons. Examples of polymers with suitable covalently attached anionicgroups (e.g., sulfonates or sulfates) includepoly(2-acrylamido-2-methyl-1-propanesulfonic acid);poly(2-acrylamido-2-methyl-1-propanesulfonic acid-co-acrylonitrile);poly(2-acrylamido-2-methyl-1-propanesulfonic acid-co-styrene);poly(vinylsulfonic acid); poly(sodium 4-styrenesulfonic acid); andsulfates and sulfonates derived from: poly(acrylic acid); poly(methylacrylate); poly(methyl methacrylate); and poly(vinyl alcohol); andpharmaceutically acceptable salts thereof. Examples ofcarbohydrate-derived polymers with suitable covalently attached anionicgroups include those of the formula:

wherein R is SO₃— or OSO₃—; and pharmaceutically acceptable saltsthereof.

Peptides and peptide derivatives can also act as carrier molecules. Theterm “peptide” includes two or more amino acids covalently attachedthrough a peptide bond. Amino acids which can be used in peptide carriermolecules include those naturally occurring amino acids found inproteins such as glycine, alanine, valine, cysteine, leucine,isoleucine, serine, threonine, methionine, glutamic acid, aspartic acid,glutamine, asparagine, lysine, arginine, proline, histidine,phenylalanine, tyrosine, and tryptophan. The term amino acid furtherincludes analogs, derivatives and congeners of naturally occurring aminoacids, one or more of which can be present in a peptide derivative. Forexample, amino acid analogs can have lengthened or shortened side chainsor variant side chains with appropriate functional groups. Also includedare the D and L stereoisomers of an amino acid when the structure of theamino acid admits of stereoisomeric forms. The term “peptide derivative”further includes compounds which contain molecules which mimic a peptidebackbone but are not amino acids (so-called peptidomimetics), such asbenzodiazepine molecules (see e.g. James, G. L. et al. (1993) Science260:1937-1942). The anionic groups can be attached to a peptide orpeptide derivative through a functional group on the side chain ofcertain amino acids or other suitable functional group. For example, asulfate or sulfonate group can be attached through the hydroxy sidechain of a serine residue. A peptide can be designed to interact with abinding site for a basement membrane constituent (e.g., HSPG) in anamyloidogenic protein (as described above). Accordingly, in oneembodiment, the peptide comprises four amino acids and anionic groups(e.g., sulfonates) are attached to the first, second and fourth aminoacid. For example, the peptide can be Ser-Ser-Y-Ser, wherein an anionicgroup is attached to the side chain of each serine residue and Y is anyamino acid. In addition to peptides and peptide derivatives, singleamino acids can be used as carriers in the therapeutic compounds of theinvention. For example, cysteic acid, the sulfonate derivative ofcysteine, can be used.

The term “aliphatic group” is intended to include organic compoundscharacterized by straight or branched chains, typically having between 1and 22 carbon atoms. Aliphatic groups include alkyl groups, alkenylgroups and alkynyl groups. In complex structures, the chains can bebranched or cross-linked. Alkyl groups include saturated hydrocarbonshaving one or more carbon atoms, including straight-chain alkyl groupsand branched-chain alkyl groups. Such hydrocarbon moieties may besubstituted on one or more carbons with, for example, a halogen, ahydroxyl, a thiol, an amino, an alkoxy, an alkylcarboxy, an alkylthio,or a nitro group. Unless the number of carbons is otherwise specified,“lower aliphatic” as used herein means an aliphatic group, as definedabove (e.g., lower alkyl, lower alkenyl, lower alkynyl), but having fromone to six carbon atoms. Representative of such lower aliphatic groups,e.g., lower alkyl groups, are methyl, ethyl, n-propyl, isopropyl,2-chloropropyl, n-butyl, sec-butyl, 2-aminobutyl, isobutyl, tert-butyl,3-thiopentyl, and the like. As used herein, the term “amino” means —NH₂;the term “nitro” means —NO₂; the term “halogen” designates —F, —Cl, —Bror —I; the term “thiol” means SH; and the term “hydroxyl” means —OH.Thus, the term “alkylamino” as used herein means an alkyl group, asdefined above, having an amino group attached thereto. The term“alkylthio” refers to an alkyl group, as defined above, having asulfhydryl group attached thereto. The term “alkylcarboxyl” as usedherein means an alkyl group, as defined above, having a carboxyl groupattached thereto. The term “alkoxy” as used herein means an alkyl group,as defined above, having an oxygen atom, attached thereto.Representative alkoxy groups include methoxy, ethoxy, propoxy,tert-butoxy and the like. The terms “alkenyl” and “alkynyl” refer tounsaturated aliphatic groups analogous to alkyls, but which contain atleast one double or triple bond respectively.

The term “alicyclic group” is intended to include closed ring structuresof three or more carbon atoms. Alicyclic groups include cycloparaffinsor naphthenes which are saturated cyclic hydrocarbons, cycloolefinswhich are unsaturated with two or more double bonds, and cycloacetyleneswhich have a triple bond. They do not include aromatic groups. Examplesof cycloparaffins include cyclopropane, cyclohexane, and cyclopentane.Examples of cycloolefins include cyclopentadiene and cyclooctatetraene.Alicyclic groups also include fused ring structures and substitutedalicyclic groups such as alkyl substituted alicyclic groups. In theinstance of the alicyclics such substituents can further comprise alower alkyl, a lower alkenyl, a lower alkoxy, a lower alkylthio, a loweralkylamino, a lower alkylcarboxyl, a nitro, a hydroxyl, —CF₃, —CN, orthe like.

The term “heterocyclic group” is intended to include closed ringstructures in which one or more of the atoms in the ring is an elementother than carbon, for example, nitrogen, or oxygen. Heterocyclic groupscan be saturated or unsaturated and heterocyclic groups such as pyrroleand furan can have aromatic character. They include fused ringstructures such as quinoline and isoquinoline. Other examples ofheterocyclic groups include pyridine and purine. Heterocyclic groups canalso be substituted at one or more constituent atoms with, for example,a halogen, a lower alkyl, a lower alkenyl, a lower alkoxy, a loweralkylthio, a lower alkylamino, a lower alkylcarboxyl, a nitro, ahydroxyl, —CF₃, —CN, or the like.

The term “aromatic group” is intended to include unsaturated cyclichydrocarbons containing one or more rings. Aromatic groups include 5-and 6-membered single-ring groups which may include from zero to fourheteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole,oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazineand pyrimidine, and the like. The aromatic ring may be substituted atone or more ring positions with, for example, a halogen, a lower alkyl,a lower alkenyl, a lower alkoxy, a lower alkylthio, a lower alkylamino,a lower alkylcarboxyl, a nitro, a hydroxyl, —CF₃, —CN, or the like.

The therapeutic compound of the invention can be administered in apharmaceutically acceptable vehicle. As used herein “pharmaceuticallyacceptable vehicle” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like which are compatible with the activity ofthe compound and are physiologically acceptable to the subject. Anexample of a pharmaceutically acceptable vehicle is buffered normalsaline (0.15 molar NaCl). The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with thetherapeutic compound, use thereof in the compositions suitable forpharmaceutical administration is contemplated. Supplementary activecompounds can also be incorporated into the compositions.

In a preferred embodiment of the method of the invention, thetherapeutic compound administered to the subject is comprised of atleast one sulfonate group covalently attached to a carrier molecule, ora pharmaceutically acceptable salt thereof. Accordingly, the therapeuticcompound can have the formula:Q-[—SO₃ ⁻X⁺]_(n)wherein Q is a carrier molecule; X⁺ is a cationic group; and n is aninteger. Suitable carrier molecules and cationic groups are thosedescribed hereinbefore. The number of sulfonate groups (“n”) is selectedsuch that the biodistribution of the compound for an intended targetsite is not prevented while maintaining activity of the compound asdiscussed earlier. In one embodiment, n is an integer between 1 and 10.In another embodiment, n is an integer between 3 and 8. As describedearlier, therapeutic compounds with multiple sulfonate groups can havethe sulfonate groups spaced such that the compound interacts optimallywith an HSPG binding site within an amyloidogenic protein.

In preferred embodiments, the carrier molecule for a sulfonate(s) is alower aliphatic group (e.g., a lower alkyl, lower alkenyl or loweralkynyl), a heterocyclic group, a disaccharide, a polymer or a peptideor peptide derivative. Furthermore, the carrier can be substituted, e.g.with one or more amino, nitro, halogen, thiol or hydroxy groups.

Examples of suitable sulfonated polymeric therapeutic compounds includepoly(2-acrylamido-2-methyl-1-propanesulfonic acid);poly(2-acrylamido-2-methyl-1-propanesulfonic acid-co-acrylonitrile);poly(2-acrylamido-2-methyl-1-propanesulfonic acid-co-styrene);poly(vinylsulfonic acid); poly(sodium 4-styrenesulfonic acid); asulfonic acid derivative of poly(acrylic acid); a sulfonic acidderivative of poly(methyl acrylate); a sulfonic acid derivative ofpoly(methyl methacrylate); and a sulfonate derivative of poly(vinylalcohol); and pharmaceutically acceptable salts thereof.

A preferred sulfonated polymer is poly(vinylsulfonic acid) (PVS) or apharmaceutically acceptable salt thereof, preferably the sodium saltthereof. In one embodiment, PVS having a molecular weight of about800-1000 Daltons is used. PVS may be used as a mixture of stereoisomersor as a single active isomer.

A preferred sulfonated disaccharide is a fully or partially sulfonatedsucrose, or pharmaceutically acceptable salt thereof, such as sucroseoctasulfonate.

Preferred lower aliphatic sulfonated compounds for use in the inventioninclude ethanesulfonic acid; 2-aminoethanesulfonic acid (taurine);cysteic acid (3-sulfoalanine or α-amino-β-sulfopropionic acid);1-propanesulfonic acid; 1,2-ethanedisulfonic acid; 1,3-propanedisulfonicacid; 1,4-butanedisulfonic acid; 1,5-pentanedisulfonic acid; and4-hydroxybutane-1-sulfonic acid; and pharmaceutically acceptable saltsthereof.

Preferred heterocyclic sulfonated compounds include3-(N-morpholino)propanesulfonic acid; andtetrahydrothiophene-1,1-dioxide-3,4-disulfonic acid; andpharmaceutically acceptable salts thereof.

In another embodiment of the method of the invention, the therapeuticcompound administered to the subject is comprised of at least onesulfate group covalently attached to a carrier molecule, or apharmaceutically acceptable salt thereof. Accordingly, the therapeuticcompound can have the formula:Q-[—OSO₃ ⁻X⁺]_(n)wherein Q is a carrier molecule; X⁺ is a cationic group; and n is aninteger. Suitable carrier molecules and cationic groups are thosedescribed hereinbefore. The number of sulfate groups (“n”) is selectedsuch that the biodistribution of the compound for an intended targetsite is not prevented while maintaining activity of the compound asdiscussed earlier. In one embodiment, n is an integer between 1 and 10.In another embodiment, n is an integer between 3 and 8. As describedearlier, therapeutic compounds with multiple sulfate groups can have thesulfate groups spaced such that the compound interacts optimally with anHSPG binding site within an amyloidogenic protein.

In preferred embodiments, the carrier molecule for a sulfate(s) is alower aliphatic group (e.g., a lower alkyl, lower alkenyl or loweralkynyl), a disaccharide, a polymer or a peptide or peptide derivative.Furthermore, the carrier can be substituted, e.g. with one or moreamino, nitro, halogen, thiol or hydroxy groups.

Examples of suitable sulfated polymeric therapeutic compounds includepoly(2-acrylamido-2-methyl-propyl sulfuric acid);poly(2-acrylamido-2-methyl-propyl sulfuric acid-co-acrylonitrile);poly(2-acrylamido-2-methyl-propyl sulfuric acid-co-styrene);poly(vinylsulfuric acid); poly(sodium 4-styrenesulfate); a sulfatederivative of poly(acrylic acid); a sulfate derivative of poly(methylacrylate); a sulfate derivative of poly(methyl methacrylate); and asulfate derivative of poly(vinyl alcohol); and pharmaceuticallyacceptable salts thereof.

A preferred sulfated polymer is poly(vinylsulfuric acid) orpharmaceutically acceptable salt thereof.

A preferred sulfated disaccharide is sucrose octasulfate orpharmaceutically acceptable salt thereof.

Preferred lower aliphatic sulfated compounds for use in the inventioninclude ethyl sulfuric acid; 2-aminoethan-1-ol sulfuric acid; 1-propanolsulfuric acid; 1,2-ethanediol disulfuric acid; 1,3-propanedioldisulfuric acid; 1,4-butanediol disulfuric acid; 1,5-pentanedioldisulfuric acid; and 1,4-butanediol monosulfuric acid; andpharmaceutically acceptable salts thereof.

Preferred heterocyclic sulfated compounds include3-(N-morpholino)propanesulfuric acid; andtetrahydrothiophene-1,1-dioxide-3,4-diol disulfuric acid; andpharmaceutically acceptable salts thereof.

A further aspect of the invention includes pharmaceutical compositionsfor treating amyloidosis. The therapeutic compounds in the methods ofthe invention, as described hereinbefore, can be incorporated into apharmaceutical composition in an amount effective to inhibit amyloidosisin a pharmaceutically acceptable vehicle.

In one embodiment, the pharmaceutical compositions of the inventioninclude a therapeutic compound that has at least one sulfonate groupcovalently attached to a carrier molecule, or a pharmaceuticallyacceptable salt thereof, in an amount sufficient to inhibit amyloiddeposition, and a pharmaceutically acceptable vehicle. The therapeuticcomposition can have the formula:Q-[—SO₃ ⁻X⁺]_(n)wherein Q is a carrier molecule; X⁺ is a cationic group; and n is aninteger selected such that the biodistribution of the compound for anintended target site is not prevented while maintaining activity of thecompound.

In another embodiment, the pharmaceutical compositions of the inventioninclude a therapeutic compound that has at least one sulfate groupcovalently attached to a carrier molecule, or a pharmaceuticallyacceptable salt thereof, in an amount sufficient to inhibit amyloiddeposition, and a pharmaceutically acceptable vehicle. The therapeuticcompound can have the following formula:Q-[—SO₃ ⁻X⁺]_(n)wherein Q is a carrier molecule; X⁺ is a cationic group; and n is aninteger selected such that the biodistribution of the compound for anintended target site is not prevented while maintaining activity of thecompound.

The invention further contemplates the use of prodrugs which areconverted in vivo to the therapeutic compounds of the invention (see,e.g., R. B. Silverman, 1992, “The Organic Chemistry of Drug Design andDrug Action”, Academic Press, Chp. 8). Such prodrugs can be used toalter the biodistribution (e.g., to allow compounds which would nottypically cross the blood-brain barrier to cross the blood-brainbarrier) or the pharmacokinetics of the therapeutic compound. Forexample, an anionic group, e.g., a sulfate or sulfonate, can beesterified, e.g, with a methyl group or a phenyl group, to yield asulfate or sulfonate ester. When the sulfate or sulfonate ester isadministered to a subject, the ester is cleaved, enzymatically ornon-enzymatically, to reveal the anionic group. Such an ester can becyclic, e.g., a cyclic sulfate or sultone, or two or more anionicmoieties may be esterified through a linking group. In a preferredembodiment, the prodrug is a cyclic sulfate or sultone. An anionic groupcan be esterified with moieties (e.g., acyloxymethyl esters) which arecleaved to reveal an intermediate compound which subsequently decomposesto yield the active compound. In another embodiment, the prodrug is areduced form of a sulfate or sulfonate, e.g., a thiol, which is oxidizedin vivo to the therapeutic compound. Furthermore, an anionic moiety canbe esterified to a group which is actively transported in vivo, or whichis selectively taken up by target organs. The ester can be selected toallow specific targeting of the therapeutic moieties to particularorgans, as described below for carrier moieties.

Carrier molecules useful in the therapeutic compounds include carriermolecules previously described, e.g. carbohydrates, polymers, peptides,peptide derivatives, aliphatic groups, alicyclic groups, heterocyclicgroups, aromatic groups or combinations thereof. Suitable polymersinclude substituted and unsubstituted vinyl, acryl, styrene andcarbohydrate-derived polymers and copolymers and salts thereof.Preferred carrier molecules include a lower alkyl group, a heterocyclicgroup, a disaccharide, a polymer or a peptide or peptide derivative.

Carrier molecules useful in the present invention may also includemoieties which allow the therapeutic compound to be selectivelydelivered to a target organ or organs. For example, if delivery of atherapeutic compound to the brain is desired, the carrier molecule mayinclude a moiety capable of targeting the therapeutic compound to thebrain, by either active or passive transport (a “targeting moiety”).Illustratively, the carrier molecule may include a redox moiety, asdescribed in, for example, U.S. Pat. Nos. 4,540,564 and 5,389,623, bothto Bodor. These patents disclose drugs linked to dihydropyridinemoieties which can enter the brain, where they are oxidized to a chargedpyridinium species which is trapped in the brain. Thus, drug accumulatesin the brain. Many targeting moieties are known, and include, forexample, asialoglycoproteins (see, e.g. Wu, U.S. Pat. No. 5,166,320) andother ligands which are transported into cells via receptor-mediatedendocytosis (see below for further examples of targeting moieties whichmay be covalently or non-covalently bound to a carrier molecule).Furthermore, the therapeutic compounds of the invention may bind toamyloidogenic proteins in the circulation and thus be transported to thesite of action.

In one embodiment, the therapeutic compound in the pharmaceuticalcompositions is a sulfonated polymer, for examplepoly(2-acrylamido-2-methyl-1-propanesulfonic acid);poly(2-acrylamido-2-methyl-1-propanesulfonic acid-co-acrylonitrile);poly(2-acrylamido-2-methyl-1-propanesulfonic acid-co-styrene);poly(vinylsulfonic acid); poly(sodium 4-styrenesulfonic acid); asulfonate derivative of poly(acrylic acid); a sulfonate derivative ofpoly(methyl acrylate); a sulfonate derivative of poly(methylmethacrylate); and a sulfonate derivative of poly(vinyl alcohol); andpharmaceutically acceptable salts thereof.

In another embodiment, the therapeutic compound in the pharmaceuticalcompositions is a sulfated polymer, for examplepoly(2-acrylamido-2-methyl-1-propanesulfuric acid);poly(2-acrylamido-2-methyl-1-propanesulfuric acid-co-acrylonitrile);poly(2-acrylamido-2-methyl-1-propanesulfuric acid-co-styrene);poly(vinylsulfuric acid); poly(sodium 4-styrenesulfate); a sulfatederivative of poly(acrylic acid); a sulfate derivative of poly(methylacrylate); a sulfate derivative of poly(methyl methacrylate); and asulfate derivative of poly(vinyl alcohol); and pharmaceuticallyacceptable salts thereof.

Preferred therapeutic compounds for inclusion in a pharmaceuticalcomposition for treating amyloidosis of the invention includepoly(vinylsulfuric acid); poly(vinylsulfonic acid); sucrose octasulfate;a partially or fully sulfonated sucrose; ethyl sulfuric acid;ethanesulfonic acid; 2-aminoethanesulfonic acid (taurine);2-(aminoethyl)sulfuric acid; cysteic acid (3-sulfoalanine orα-amino-β-sulfopropionic acid); 1-propanesulfonic acid; propyl sulfuricacid; 1,2-ethanedisulfonic acid; 1,2-ethanediol disulfuric acid;1,3-propanedisulfonic acid; 1,3-propanediol disulfuric acid;1,4-butanedisulfonic acid; 1,4-butanediol disulfuric acid;1,5-pentanedisulfonic acid; 1,5-pentanediol disulfuric acid;4-hydroxybutane-1-sulfonic acid;tetrahydrothiophene-1,1-dioxide-3,4-disulfonic acid;3-(N-morpholino)propanesulfonic acid; and pharmaceutically acceptablesalts thereof.

In the methods of the invention, amyloid deposition in a subject isinhibited by administering a therapeutic compound of the invention tothe subject. The term subject is intended to include living organisms inwhich amyloidosis can occur. Examples of subjects include humans,monkeys, cows, sheep, goats, dogs, cats, mice, rats, and transgenicspecies thereof. Administration of the compositions of the presentinvention to a subject to be treated can be carried out using knownprocedures, at dosages and for periods of time effective to inhibitamyloid deposition in the subject. An effective amount of thetherapeutic compound necessary to achieve a therapeutic effect may varyaccording to factors such as the amount of amyloid already deposited atthe clinical site in the subject, the age, sex, and weight of thesubject, and the ability of the therapeutic compound to inhibit amyloiddeposition in the subject. Dosage regimens can be adjusted to providethe optimum therapeutic response. For example, several divided doses maybe administered daily or the dose may be proportionally reduced asindicated by the exigencies of the therapeutic situation. A non-limitingexample of an effective dose range for a therapeutic compound of theinvention (e.g., poly(vinylsulfonate sodium salt)) is between 5 and 500mg/kg of body weight/per day. In an aqueous composition, preferredconcentrations for the active compound (i.e., the therapeutic compoundthat can inhibit amyloid deposition) are between 5 and 500 mM, morepreferably between 10 and 100 mM, and still more preferably between 20and 50 mM. For taurine, particularly preferred aqueous concentrationsare between 10 and 20 mM.

As demonstrated in the Exemplification, the therapeutic compounds of theinvention are effective when administered orally. Accordingly, apreferred route of administration is oral administration. Alternatively,the active compound may be administered by other suitable routes suchsubcutaneous, intravenous, intraperitoneal, etc. administration (e.g. byinjection). Depending on the route of administration, the activecompound may be coated in a material to protect the compound from theaction of acids and other natural conditions which may inactivate thecompound.

The compounds of the invention can be formulated to ensure properdistribution in vivo. For example, the blood-brain barrier (BBB)excludes many highly hydrophilic compounds. To ensure that thetherapeutic compounds of the invention cross the BBB, they can beformulated, for example, in liposomes. For methods of manufacturingliposomes, see, e.g., U.S. Pat. Nos. 4,522,811; 5,374,548; and5,399,331. The liposomes may comprise one or more moieties which areselectively transported into specific cells or organs (“targetingmoieties”), thus providing targeted drug delivery (see, e.g., V. V.Ranade (1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moietiesinclude folate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low etal.); mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun.153:1038); antibodies (P. G. Bloeman et al. (1995) FEBS Lett. 357:140;M. Owais et al. (1995) Antimicrob. Agents Chemother. 39: 180);surfactant protein A receptor (Briscoe et al. (1995) Am. J. Physiol.1233:134); gp120 (Schreier et al. (1994) J. Biol. Chem. 269:9090); seealso K. Keinanen; M. L. Laukkanen (1994) FEBS Lett. 346: 123; J. J.Killion; I. J. Fidler (1994) Immunomethods 4:273. In a preferredembodiment, the therapeutic compounds of the invention are formulated inliposomes; in a more preferred embodiment, the liposomes include atargeting moiety.

To administer the therapeutic compound by other than parenteraladministration, it may be necessary to coat the compound with, orco-administer the compound with, a material to prevent its inactivation.For example, the therapeutic compound may be administered to a subjectin an appropriate carrier, for example, liposomes, or a diluent.Pharmaceutically acceptable diluents include saline and aqueous buffersolutions. Liposomes include water-in-oil-in-water CGF emulsions as wellas conventional liposomes (Strejan et al., (1984) J. Neuroimmunol.7:27).

The therapeutic compound may also be administered parenterally,intraperitoneally, intraspinally, or intracerebrally. Dispersions can beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations may contain a preservative to prevent the growth ofmicroorganisms.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. In all cases, the composition must be sterileand must be fluid to the extent that easy syringability exists. It mustbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms such asbacteria and fungi. The vehicle can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and vegetable oils. The proper fluidity canbe maintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, ascorbic acid,thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, sodium chloride, orpolyalcohols such as mannitol and sorbitol, in the composition.Prolonged absorption of the injectable compositions can be brought aboutby including in the composition an agent which delays absorption, forexample, aluminum monostearate or gelatin.

Sterile injectable solutions can be prepared by incorporating thetherapeutic compound in the required amount in an appropriate solventwith one or a combination of ingredients enumerated above, as required,followed by filtered sterilization. Generally, dispersions are preparedby incorporating the therapeutic compound into a sterile vehicle whichcontains a basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and freeze-drying which yields a powder ofthe active ingredient (i.e., the therapeutic compound) plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The therapeutic compound can be orally administered, for example, withan inert diluent or an assimilable edible carrier. The therapeuticcompound and other ingredients may also be enclosed in a hard or softshell gelatin capsule, compressed into tablets, or incorporated directlyinto the subject's diet. For oral therapeutic administration, thetherapeutic compound may be incorporated with excipients and used in theform of ingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. The percentage of thetherapeutic compound in the compositions and preparations may, ofcourse, be varied. The amount of the therapeutic compound in suchtherapeutically useful compositions is such that a suitable dosage willbe obtained.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subjects to be treated; each unitcontaining a predetermined quantity of therapeutic compound calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical vehicle. The specification for the dosage unitforms of the invention are dictated by and directly dependent on (a) theunique characteristics of the therapeutic compound and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such a therapeutic compound for the treatment ofamyloid deposition in subjects.

Active compounds are administered at a therapeutically effective dosagesufficient to inhibit amyloid deposition in a subject. A“therapeutically effective dosage” preferably inhibits amyloiddeposition by at least about 20%, more preferably by at least about 40%,even more preferably by at least about 60%, and still more preferably byat least about 80% relative to untreated subjects. The ability of acompound to inhibit amyloid deposition can be evaluated in an animalmodel system that may be predictive of efficacy in inhibiting amyloiddeposition in human diseases, such as the model system used in theExamples. Alternatively, the ability of a compound to inhibit amyloiddeposition can be evaluated by examining the ability of the compound toinhibit an interaction between an amyloidogenic protein and a basementmembrane constituent, e.g., using a binding assay such as that describedhereinbefore.

The method of the invention is useful for treating amyloidosisassociated with any disease in which amyloid deposition occurs.Clinically, amyloidosis can be primary, secondary, familial or isolated.Amyloids have been categorized by the type of amyloidogenic proteincontained within the amyloid. Non-limiting examples of amyloids whichcan be inhibited, as identified by their amyloidogenic protein, are asfollows (with the associated disease in parentheses after theamyloidogenic protein): β-amyloid (Alzheimer's disease, Down's syndrome,hereditary cerebral hemorrhage amyloidosis [Dutch]); amyloid A (reactive[secondary] amyloidosis, familial Mediterranean Fever, familial amyloidnephropathy with urticaria and deafness [Muckle-Wells syndrome]);amyloid κL-chain or amyloid λL-chain (idiopathic [primary], myeloma ormacroglobulinemia-associated); Aβ2M (chronic hemodialysis); ATTR(familial amyloid polyneuropathy [Portuguese, Japanese, Swedish],familial amyloid cardiomyopathy [Danish], isolated cardiac amyloid,systemic senile amyloidosis); AIAPP or amylin (adult onset diabetes,insulinoma); atrial naturetic factor (isolated atrial amyloid);procalcitonin (medullary carcinoma of the thyroid); gelsolin (familialamyloidosis [Finnish]); cystatin C (hereditary cerebral hemorrhage withamyloidosis [Icelandic]); AApoA-I (familial amyloidotic polyneuropathy[Iowa]); AApoA-II (accelerated senescence in mice);fibrinogen-associated amyloid; lysozyme-associated amyloid; and AScr orPrP-27 (Scrapie, Creutzfeldt-Jacob disease,Gerstmann-Straussler-Scheinker syndrome, bovine spongiformencephalitis).

The sulfated and sulfonated compounds used in the methods describedherein are commercially available (e.g. Sigma Chemical Co., St. Louis,Mo., or Aldrich Chemical Co., Milwaukee, Wis.) and/or can be synthesizedby standard techniques known in the art (see, e.g., Stone, G. C. H.(1936) J. Am. Chem. Soc., 58:488). In general, sulfated compounds weresynthesized from the corresponding alcohols. The alcohols correspondingto WAS-28 and WAS-29 were obtained by reduction of1,3-acetonedicarboxylic acid and triethyl methanetricarboxylate,respectively, which are commercially available. Representative synthesesof active compounds used herein are described in further detail inExample 10.

In certain embodiments of the invention, Congo red is excluded fromsulfonated compounds used in the method of the invention.

In certain embodiments of the invention, the following sulfatedcompounds are excluded from use in the method of the invention: dextransulfate 500, ι-carrageenan, λ-carrageenan, dextran sulfate 8,κ-carrageenan, pentosan polysulfate, and/or heparan.

In certain embodiments of the invention, the compositions and methods ofthe invention are used to inhibit amyloid deposition in amyloidosiswherein the amyloidogenic protein is not the protease-resistant form ofa prion protein, AScr (also known as PrP-27).

The invention is further illustrated by the following examples whichshould not be construed as further limiting the subject invention. Thecontents of all references, issued patents, and published patentapplications cited throughout this application are hereby incorporatedby reference. A demonstration of efficacy of the therapeutic compoundsof the present invention in the mouse model described in the examples ispredictive of efficacy in humans.

EXEMPLIFICATION

In the following examples, a well-characterized mouse model ofamyloidosis was used. In this in vivo system, animals receive aninflammatory stimulus and amyloid enhancing factor. For acuteamyloidosis (i.e., short term amyloid deposition), the inflammatorystimulus is AgNO₃. For chronic amyloidosis (ongoing amyloid deposition),the inflammatory stimulus is lipopolysaccharide (LPS). Amyloiddeposition (AA amyloid) in the spleens of mice was measured with andwithout therapeutic treatment.

Example 1

The following methodologies were used:

Animals

All mice were of the CD strain (Charles Rivers, Montreal, Quebec) andweighing 25-30 g.

Animal Treatment

All animals received AgNO₃ (0.5 ml, 2% solution) subcutaneously in theback, and amyloid enhancing factor (AEF) 100 μg intravenously. Thepreparation of amyloid enhancing factor has been described previously inAxelrad, M. A. et al. (“Further Characterization of Amyloid EnhancingFactor” Lab. Invest. 47:139-146 (1982)). The animals were divided intoseveral groups one of which was an untreated control group which wassacrificed six days later. The remaining animals were divided into thosewhich received poly(vinylsulfonate sodium salt) (PVS) at 50 mg, 40 mg,20 mg, or 10 mg by intraperitoneal injection every 12 hours or sucroseoctasulfate ammonium salt (SOA) at 73 mg or 36.5 mg every 8 hours by IPinjection. The PVS used in this and all subsequent Examples was amixture of stereoisomers. Surviving animals were sacrificed on the 5thday of treatment. In all cases the PVS or SOA was dissolved in a sterileaqueous carrier.

Tissue Preparation

At the termination of the experiments, the animals were sacrificed bycervical dislocation and the spleens, livers, and kidneys were fixed in96% ethanol, 1% glacial acetic acid and 3% water as described in Lyon,A. W. et al. (“Co-deposition of Basement Membrane Components During theInduction of Murine Splenic AA Amyloid” Lab. Invest. 64:785-790 (1991)).Following fixation, the tissues were embedded in paraffin, 8-10 micronsections were cut and stained with Congo Red without counterstain asdescribed in Puchtler, H. et al. (“Application of Thiazole Dyes toAmyloid Under Conditions of Direct Cotton Dyeing: Correlation ofHistochemical and Chemical Data” Histochemistry 77:431-445 (1983)). Thehistologic sections viewed under polarized light were assessed by imageanalysis for the percent of spleen occupied by amyloid. In the case ofthe experiments with sucrose octasulfate, the tissues were immunostainedwith an antibody to the SAA protein (described in Lyon A. W. et al. LabInvest. 64:785-790 (1991)) and the immunostained sections assessed byimage analysis for the percent of tissue section occupied by amyloid.

Viability of Animals

All control animals survived the experiment without incident. In thecase of the animals undergoing therapy, all animals given sucroseoctasulfate at 73 mg/injection succumbed prior to the termination of theexperiment. Animals receiving 36.5 mg of sucrose octasulfate/injectionall survived. Of those animals receiving PVS (molecular weight 900-1000)in each dosage group, approximately half to one-third of the animalssuccumbed prior to the termination of the experiment. In all cases ofanimal deaths prior to the end of the experiments, the cause of deathwas uncontrolled intraperitoneal hemorrhage.

Effects of Agents on Amyloid Deposition

The effect of sucrose octasulfate at 36.5 mg/injection is shown below inTable 1. The mean area of spleen occupied by amyloid in control animalswas 7.8%±1.5% S.E.M. In animals receiving the therapeutic agent the meanarea was 3.2%±0.5% S.E.M. The difference is significant at a p≦0.02.TABLE 1 Effect of Sucrose Octasulfate Ammonium Salt on AA AmyloidDeposition In vivo in Mouse Spleen % Area Occupied by Amyloid Untreated7.8 + 1.5 n = 5 Sucrose OctaS0₄ 3.2 + 0.5 n = 5 p ≦ 0.02

In the case of PVS, the data are shown in FIG. 1. There was a profoundinhibition of amyloid deposition at all doses with the suggestion of adose-dependent effect. An effective dose range is between 5 and 500mg/kg of body weight/per day.

Preliminary assessment of the plasma level of the precursor ofinflammation-associated amyloidosis, SAA, has shown that there is nodifference between the animals being treated with PVS and thoseuntreated.

The method of administering the agents of the present invention isbelieved to have had an effect upon the mortality rate of the animals.Intraperitoneal injection was selected as providing a large membranesurface for ease of access to the circulating system. However, likeheparan, the compounds of the present invention exhibit anti-coagulantproperties. Repeated injections through the peritoneal wall inducedsevere hemorrhaging and ultimately resulted in filling the peritonealcavity, with loss of blood causing death. While subcutaneous injectionwould result in slower absorption of the active compound, it is lesslikely that this route would cause hemorrhaging to such an extent as tocause death. Oral administration of the compounds was performed insubsequent experiments (see below).

Example 2

Swiss white mice weighing 25-30 g were given Amyloid Enhancing Factor(AEF) and AgNO₃ as described previously (Kisilevsky, R. and Boudreau, L.(1983) “The kinetics of amyloid deposition: I. The effect of amyloidenhancing factor and splenectomy” Lab. Invest., 48, 53-59), to induceamyloidosis. Twenty four (24) hours later they were divided into threegroups. One group served as a control and was maintained on standardlaboratory mouse chow and tap water ad lib. A second group received thestandard chow but its water contained 20 mg/ml of poly(vinylsulfonatesodium salt) (PVS). The third group had 50 mg/ml of PVS in its drinkingwater. Fluid intake in both groups was the same. All animals weresacrificed on day six (6) of the experiment, their spleens collected,prepared for sectioning, spleen sections stained with Congo red(Puchtler, H., et al. (1983) “Application of Thiazole Dyes to Amyloidunder Conditions of Direct Cotton Dyeing: Correlation of Histochemicaland Chemical Data” Histochemistry, 77, 431445), and the percent areaoccupied by amyloid assessed by an image analysis apparatus and program(MCID M2, Imaging Research Inc., Brock University, St. Catherines,Ontario, Canada). As shown in FIG. 4, oral administration of PVSinterferes with amyloid deposition in a dose dependent manner.

Example 3

Since it was possible that PVS was inhibiting the hepatic synthesis ofthe amyloid precursor, and thus failure to deposit amyloid was due tothe absence of the precursor pool, the effect of PVS on the blood levelof the amyloid precursor (SAA) during the course of the experiment wasdetermined. Animals received AEF+AgNO₃ as described above and weredivided into two groups. Group 1 received no further treatment. Twentyfour hours later, Group 2 received 50 mg of PVS by intraperitonealinjection every 12 hours for a period of 5 days. To plot the level ofSAA during this process, each animal (controls and experimentals) wasbled from the tail (≈25 μl) each day. The SAA levels in these sampleswere determined by a solid phase ELISA procedure (described inBrissette, L., et al. (1989) J. Biol. Chem., 264, 19327-19332). Theresults are shown in FIG. 5. The open circles represent the data fromthe PVS-treated mice, while the triangles show the data from thenon-treated animals. SAA levels were equivalent in treated and untreatedanimals, demonstrating that PVS does not mediate its effect bypreventing the synthesis of SAA.

Example 4

In the above described experiments, therapy with PVS was begun 24 hoursinto the amyloid induction protocol. This does not mimic a clinicalsituation where the patient usually has well established amyloid. Toapproximate a more realistic clinical situation, a separate set ofexperiments were performed in which PVS treatment was begun afteramyloid deposition had already begun. Animals received AEF+AgNO₃, asdescribed above, remained on tap water for 7 days, after which they wereseparated into two groups. Group 1 remained on standard food and tapwater. Group 2 remained on standard food but had 50 mg/ml of PVS addedto their drinking water. To assess the effect of PVS on the course ofamyloid deposition after amyloid was already present, five animals ineach group were sacrificed on days 7, 10, 14, and 17. The spleens wereprocessed and evaluated as described above. The data are shown in FIG.6. Control animals (triangles) continued to deposit amyloid for 14 days,following which the quantity of amyloid began to decrease. This latterdecrease is most likely due to the fact that only one injection ofAgNO₃, the inflammatory stimulus, was given and, after 14 days, the SAAlevels are known to decrease (Kisilevsky, R., Boudreau, L. and Foster,D. (1983) “Kinetics of amyloid deposition. II. The effects ofdimethylsulfoxide and colchicine therapy” Lab. Invest., 48, 60-67). Inthe absence of precursor, further amyloid cannot be deposited andexisting deposits are mobilized (Kisilevsky, R. and Boudreau, L. (1983)“The kinetics of amyloid deposition: I. The effect of amyloid enhancingfactor and splenectomy” Lab. Invest., 48, 53-59). In contrast, thetreated group of animals (open circles) stopped deposition of amyloidwithin 3 days of being placed on PVS. This demonstrates that PVS iseffective at inhibiting ongoing deposition of amyloid.

Example 5

To maintain the inflammation and the blood SAA levels, and allow amyloidto be continuously deposited for the duration of a longer termexperiment, the nature of the inflammatory stimulus was changed. So asto maintain the inflammation, animals received lipopolysaccharide (LPS,20 μg)+AEF on day 0 and LPS was given by intraperitoneal injection every2nd day. On day seven (7), the animals were separated into two groups asdescribed in Example 4. Assessment of amyloid over the course of theexperiment proceeded as described in Example 4. The data are shown inFIG. 7. The control group (triangles) continued to deposit amyloid forthe entire 17 day period. Those receiving PVS apparently stoppeddepositing amyloid by day 14 (open circles and dashed line). The data onday 17 represent 4 animals per group as one animal was omitted from thistime period. The quantity of amyloid in this particular individual wasso far removed from all other data points (treated or not, it was 21%)that it is believed that this was a statistically valid procedure. Ifthis individual is included, the curve is represented by the dotted lineand the remaining open circle. It should be pointed out that animalsreceiving PVS began to develop a significant diarrhea as the experimentproceeded.

Example 6

In this experiment, another sulfonated compound, ethane monosulfonicacid was used to inhibit amyloidosis. Ethane monosulfonic acid, sodiumsalt, (EMS) is structurally the monomeric unit of PVS. Animals weregiven LPS+AEF as in Experiment 5, but on day seven EMS was used in thedrinking water as the therapeutic agent. On day seven, the animals weredivided into three groups. Group 1 was the untreated group. Group 2received 2.5 mg/ml EMS in their drinking water. Group 3 received 6 mg/mlin their drinking water. Animals were sacrificed on days 7, 10, 14, and17. These animals did not develop gastro-intestinal problems. These dataare shown in FIG. 8. Animals receiving 6 mg/ml EMS in their drinkingwater (open squares) stopped depositing amyloid after day 14. Thosereceiving 2.5 mg/ml EMS (open circles) seemed to have an abortivetherapeutic effect, with a slight diminution in the rate of amyloiddeposition at day 14 which was not maintained by day 17.

Example 7

The Influence of PVS on HSPG Binding to the Alzheimer's AmyloidPrecursor Protein (Beta APP)

The binding of heparan sulfate proteoglycan to beta APP was assessedusing an enzyme-linked immunosorbent assay technique as described inNarindrasorasak, S. et al. (“High Affinity Interactions Between theAlzheimer's Beta-Amyloid Precursor Proteins and the Basement MembraneForm of Heparan Sulfate Proteoglycan” J. Biol. Chem. 266:12878-12883(1991)). Polystyrene microtiter plates (Linbro, Flow Laboratories) werecoated with a 100 μl solution, 1 μg/ml of β-APP, in 20 mM NaHCO₃ buffer,pH 9.6. After overnight incubation at 4° C., the plates were rinsed with0.15 M NaCl, 20 mM Tris-Cl, pH 7.5 (TBS). The plates were then incubatedwith 150 μl of 1% bovine serum albumin (BSA) in TBS for 2 hours at 37°C. to block the residual hydrophobic surface on the wells. After rinsingwith TBS containing 0.05% (w/v) Tween 20 (TBS-Tween), 100 μl of variousconcentrations of HSPG in TBS-Tween were added alone or 500 μg/ml ofPVS, either in Tris-buffered saline (TBS) or phosphate-buffered saline(PBS), was included in the binding assay to assess the effect of PVS onHSPG binding to β-APP. The plates were left overnight at 4° C. to permitmaximum binding of HSPG to β-APP. The plates were then washedextensively and incubated 2 hours at 37° C. with 100 μl of anti-HSPGdiluted in TBS-Tween containing 0.1% BSA. The plates were washed againand incubated for another 2 hours with 100 μl of goat anti-rabbit IgGconjugated with alkaline phosphatase (1:2000 dilution) in TBS-Tweencontaining BSA as above. Finally, after further washing, the boundantibodies were detected by adding an alkaline phosphatase substratesolution (100 μl) containing 2 mg/ml p-nitrophenyl phosphate, 0.1 mMZnCl₂, 1 mM MgCl₂, and 100 mM glycine, pH 10. The plates were left atroom temperature for 15-120 minutes. The enzyme reaction was stopped byaddition of 50 μl of 2 M NaOH. The absorbence of the releasedp-nitrophenol was measured at 405 nm with a Titertek Multiscan/MCC 340(Flow Laboratories). The amounts of HSPG bound were determined by thenet A₄₀₅ after subtracting the A from blank wells in which the HSPGincubation step was omitted. The effect of PVS on HSPG: beta-APP bindingis illustrated in FIG. 2 (in TBS) and FIG. 3 (in PBS). Approximately30-50% inhibition of binding is demonstrated with this compound.

Example 8

Acute amyloidosis was elicited in mice with AgNO₃ and amyloid enhancingfactor as described in Examples 1 and 2. Twenty-four hours later, theanimals were divided into a control group and six test groups. Thecontrol group was maintained on standard laboratory mouse chow and tapwater ad lib. The test groups received standard chow but their watercontained 50 mM of one of the following six compounds: sodiumethanesulfonate, sodium 2-aminoethanesulfonate (taurine), sodium1-propanesulfonate, sodium 1,2-ethanedisulfonate, sodium1,3-propanedisulfonate, or sodium 1,4-butanedisulfonate. Water intakewas approximately equivalent for all groups. After six days, the animalswere sacrificed and their spleens were processed as described in Example2. For preliminary analysis, the spleen sections were examined visuallyunder a microscope for differences in amyloid deposition in the treatedanimals versus the control animals.

The results indicated that animals treated with sodium1-propanesulfonate, sodium 1,2-ethanedisulfonate, or sodium1,3-propanedisulfonate had less amyloid deposition than control animals.Under the conditions used in this experiment, animals treated withsodium ethanesulfonate, taurine sodium salt, or sodium1,4-butanedisulfonate were not observed to have less amyloid depositionthan control animals. However, these compounds may exhibit effectivenessunder other conditions, for example sodium ethanesulfonate has beenobserved to inhibit chronic amyloid deposition (see Example 6) andtaurine inhibits acute amyloid deposition at other concentrations (seeExample 9).

This experiment suggests that oral administration of sulfonated loweraliphatics such as sodium 1-propanesulfonate, sodium1,2-ethanedisulfonate and sodium 1,3-propanedisulfonate can inhibitamyloid deposition in an acute amyloidogenic system.

Example 9

In view of the preliminary results described in Example 8, furtherexperiments were conducted to determine the effect of a panel ofsulfated or sulfonated compounds on acute amyloid deposition. Acuteamyloidosis was induced in mice as described in Examples 1 and 2.Twenty-four hours later, the animals were divided into a control groupand test groups. The control group was maintained on standard laboratorymouse chow and tap water ad lib. The test groups received standard chowbut their water contained 20 or 50 mM of one of the compounds listed inTable 2, below (the chemical structures of the WAS compounds listed inTable 2 are depicted in FIGS. 9 and 10). One compound, taurine, wastested at concentrations of 5 mM, 20 mM, and 50 mM. All compounds weredissolved in water containing 1.0% sucrose. Water intake wasapproximately equivalent for all groups. After six days, the animalswere sacrificed and their spleens were processed as described in Example2.

The results are summarized in Table 2, below. TABLE 2 Effect of Sulfatedand Sulfonated Compounds on AA Amyloid Deposition In vivo in MouseSpleen Concentration Amyloid Standard Compound (mM) Deposition* Error1,5-Pentanedisulfonate^(†) 50 76 11 20 60 20 1,6-Hexanedisulfonate^(†)50 117 17 20 98 26 1,2-Ethanediol disulfate^(†) 50 8 2 20 36 101,3-Propanediol disulfate^(†) 50 11 4 20 32 11 1,4-Butanedioldisulfate^(†) 50 54 22 20 44 11 Taurine 50 68 15 20 45 23 10 34 16 5 9533 WAS-10 50 79 22 20 80 23 WAS-11 50 114 20 114 WAS-12 50 55 20 74WAS-13 50 81 20 63 WAS-14 50 135 27 20 83 28 WAS-15 50 56 13 20 102 24WAS-16 50 48 12 20 98 30 WAS-17 50 60 21 20 54 31 WAS-18 50 110 35 20 9750 WAS-19 50 61 13 20 117 28 WAS-20 50 192 37 20 119 19 WAS-21 50 158 1920 130 28 WAS-22 50 83 19 20 155 28 WAS-23 50 66 12 20 94 11 WAS-24 50103 19 20 110 15 WAS-27 50 100 18 20 86 30 WAS-28 50 56 20 53 WAS-34 5053 20 59 WAS-35 50 51 WAS-36 50 71 WAS-37 50 100 20 102 WAS-38 50 81^(†)As the sodium salt.*Amyloid deposition is given as a percentage of untreated control. Allmeasurements are the average of 3-5 animals.

The results indicate that animals treated with sodium 1,2-ethanedioldisulfate or sodium 1,3-propanediol disulfate had at least about a 65%decrease in amyloid deposition at 20 mM and at least about a 90%decrease in amyloid deposition at 50 mM. Animals treated with sodium1,4-butanediol disulfate (50 mM), sodium 1,5-pentanedisulfonate ((50mM), taurine (sodium 2-amino-ethanesulfonate) (10-20 mM),3-(cyclohexylamino)-1-propane sulfonate (WAS-12) (50 mM),4-(2-hydroxyethyl)-1-piperazine-ethanesulfonate (WAS-13) (20 mM),3-(N-morpholino)propanesulfonic acid (MOPS) (WAS-15) or its sodium salt(WAS-16) (50 mM), sodium tetrahydrothiophene-1,1-dioxide-3,4-disulfatetrihydrate (WAS-19), sodium 4-hydroxybutane-1-sulfonate (WAS-17) (50mM), sodium 1,3,5-pentanetriol trisulfate (WAS-28) (20 and 50 mM),2-aminoethyl hydrogen sulfate (WAS-34) (20 and 50 mM), indigo carmine(WAS-35) (50 mM) had at least approximately a 40% decrease in amyloiddeposition compared to untreated control animals. Taurine was effectiveat concentrations of 10-20 mM, as seen in this example, but lesseffective at 5 mM or 50 mM (see also Example 8).

Certain sulfated or sulfonated compounds were not effective in reducingthe amount of amyloid deposition under the conditions employed, but maybe effective in other embodiments. Earlier in vitro work demonstratedthat dermatan sulfate and chondroitin 6-sulfate do not interfere withthe binding of beta amyoid presursor protein to HSPG.Sodium(±)-10-camphorsulfonate (WAS-22),4,5-dihydroxy-1,3-benzenedisulfonic acid, disodium salt (WAS-2 1) and2,5-dihydroxy-1,4-benzenedisulfonic acid, dipotassium salt (WAS-20) weretested in the above-described mouse model and, as shown in Table 2, werefound not to reduce amyloid deposition.

Example 10

In this example representative syntheses of two compounds used in themethods of the invention are described.

Sodium ethane-1,2-disulfonate

A mixture of 1,2-dibromoethane (37.6 g, 0.20 mol) and sodium sulfite(63.0 g, 0.5 mol) in water (225 mL) was heated at reflux temperature for20 h. After the mixture was cooled in the refrigerator, crystals werecollected. The crude product was repeatedly recrystallized fromwater-ethanol. The trace amount of inorganic salts was removed bytreating the aqueous solution with a small amount of silver(I) oxide andbarium hydroxide. The basic solution was neutralized with Amberlite-120ion-exchange resin and treated three times with Amberlite-120 (sodiumform) ion-exchange resin. After removal of the water, the product wasrecrystallized from water-ethanol to afford the title compound (30.5 g).

Sodium 1,3-propanedisulfonate

This compound was prepared by a modification of the method described inStone, G. C. H. (1936) J. Am. Chem. Soc., 58:488. 1,3-Dibromopropane(40.4 g, 0.20 mol) was treated with sodium sulfite (60.3 g, 0.50 mol) inwater at reflux temperature for 48 h. Inorganic salts (sodium bromideand sodium sulfite) were removed by successive treatment of theresultant reaction mixture with barium hydroxide and silver(I)oxide. Thesolution was then neutralized with Amberlite-120 (acid form) anddecolorized with Norit-A. Barium ions were removed by treatment of theaqueous solution with Amberlite-120 (sodium form) ion-exchange resin.The solvent was removed on a rotary evaporator, and the crude productwas recrystallized from water-ethanol several times to give the titlecompound (42.5 g). The small amount of trapped ethanol was removed bydissolving the crystals in a minimum amount of water and thenconcentrating the solution to dryness. The pure product was furtherdried under high vacuum at 56° C. for 24 h: mp>300° C.; ¹H NMR (D₂O), δ:3.06-3.13 (m, 4H, H-1 and H-3), 2.13-2.29 (m, 2H, H-2); ¹³C NMR (D₂O),δ: 52.3 (C-1 and C-3), 23.8 (C-2).

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific procedures described herein. Such equivalents are considered tobe within the scope of this invention and are covered by the followingclaims.

1. A method for treating amyloidosis in a subject comprising orallyadministering to the subject an effective amount of a therapeuticcompound, the therapeutic compound comprising at least one sulfonategroup covalently attached to a carrier molecule, or a pharmaceuticallyacceptable salt thereof.
 2. A compound selected from the groupconsisting of:

and pharmaceutically acceptable salts thereof.
 3. The compound of claim1, wherein the pharmaceutically acceptable salt is a sodium salt.
 4. Apharmaceutical composition comprising an effective amount of thecompound of claim 1, and a pharmaceutically acceptable vehicle.
 5. Thepharmaceutical composition of claim 4, wherein said composition isformulated for oral administration.
 6. The pharmaceutical composition ofclaim 4, wherein said composition is formulated for the treatment ofamyloidosis.
 7. A method for the prevention or treatment of anamyloid-related disease, comprising administering the compound of claim2 to a subject.
 8. The method of claim 7, wherein the subject is ahuman.
 9. The method of claim 7, wherein the compound is administeredorally.
 10. A method for inhibiting amyloid deposition in a subject,comprising administering to the subject an effective amount of thecompound of claim 2.